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Flow & Imaging Cytometry Core Facility

流动

基本信息

批准号：
10708657
负责人：
Dragan Maric
金额：
$ 74.93万
依托单位：
NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10708657
关键词：
Apoptotic Basic Science Basophils Biological Biological Assay Biological Markers Bone Marrow Calcium Calibration Cell Culture Techniques Cell Death Cell Differentiation process Cell Nucleus Cell Proliferation Cell Separation Cell membrane Cell surface Cells Cellular Assay Cellular biology Cerebrospinal Fluid Clinical Research Color Complex Core Facility Custom Cytometry Cytoplasm Data Data Analyses Detection Dyes Eligibility Determination Endothelial Cells Equipment Flow Cytometry Fluorescence-Activated Cell Sorting Gene Expression Genes Hematopoietic Human Resources Image Image Cytometry Imaging Techniques Immunoprecipitation In Situ In Vitro Individual Institutes Journals Laboratories Leukocytes Link Logic Lymphocyte Maintenance Manuscripts Mesenchymal MicroRNAs Microglia Mission Mitochondria Molecular National Institute of Neurological Disorders and Stroke Necrosis Neuroglia Neurons Nuclear Protein Peer Review Phenotype Physiological Population Preparation Property Proteins Protocols documentation Publications Quality Control RNA Reporter Research Research Personnel Research Project Grants Research Support Resources Reverse Transcriptase Polymerase Chain Reaction Sampling Services Signal Transduction Small Interfering RNA Sorting Source Specific qualifier value Spleen Stains Standardization Students Synaptosomes Testing Time Tissues Training Transfection Translational Research Transplantation United States National Institutes of Health Western Blotting Work base cell preparation cell type cytotoxic data acquisition design eosinophil experimental study high throughput screening in vivo insight instrument interest investigator training loss of function mast cell monocyte multidisciplinary nerve stem cell neutrophil novel peripheral blood programs protein expression research and development specific biomarkers stem cells transcriptome sequencing

项目摘要

NINDS Flow and Imaging Cytometry Core Facility (Facility) was established in 2001 by NINDS DIR as a crucial resource to NINDS and other intramural investigators to support their work in both basic and clinical research programs. The Facility continuously provides seamless use of flow cytometry and imaging equipment managed by the Facility as well as the crucial expertise to train investigators in proper and optimal use of this equipment. The Facility staff is also available to provide constructive input in designing and optimization of a variety of novel and customized multiplex staining protocols using unique combinations of appropriate cell surface, cytoplasmic, nuclear, protein, gene expression and physiological indicator dye biomarkers that are of specific interest to initiating investigator(s) for any given project. When requested, the Facility staff routinely carry out multi-color flow and in situ cytometry experiments and analyze the data from single or multiple cell populations of interest. Applying user-defined Boolean logic gating strategies based on different combinations of biomarkers tested, the Facility provides quantitative accounts and interpretations of the complex signal distributions in individual cells or cell populations of interest to each investigator. Using the preparative cell sorting capability of flow cytometers, the Facility staff is available to assists with isolation of highly purified cell populations and subpopulations from heterogeneous cell preparations, as requested by the initiating investigator. These include sorting of neural stem and progenitor cells, specific neuronal and glial cell types, as well as non-neural cell types (microglia, endothelial cells) from both CNS and PNS tissues. In addition, the Facility routinely sorts non-CNS-derived stem cells (hematopoietic, mesenchymal and Hoechst dye-excluding side populations) and different leukocyte phenotypes (lymphocytes, monocytes, neutrophils, basophils, eosinophils, mast cells) from the bone marrow, peripheral blood, spleen, and cerebrospinal fluid. These highly-purified cells of interest are then routinely used by NINDS and other intramural investigators for further in vitro and in vivo studies including cell culture, transplantation, time-lapse and physiological indicator dye imaging, and for transduction or transfection with specific gene constructs of interest. Additionally, FACS-purified cells have been utilized as an invaluable source of proteins for Western blotting and immunoprecipitation experiments as well as a source of RNA for RT-PCR, microarray, RNA-Seq, and microRNA assays. These cells have also provided unprecedented insights into specific loss-of-function experiments using antisense and siRNA probes. The major significance in using FACS-purified homogeneous cells in the abovementioned applications is that this approach reduces the ambiguity in the findings inherently present when studying heterogeneous cell preparations. Taken together, the routine applications of quantitative flow and in situ cytometry assays and preparative cell sorting services provided by this Facility has produced numerous insights into cell biology for NINDS and other intramural investigators, which could not otherwise be demonstrated. Since inception in 2001, the Facility has provided these invaluable services to more than 500 NINDS and other intramural investigators, fellows, students, and scientific staff and supported the multidisciplinary scientific research in over 700 basic, translational, and clinical research projects from 105 laboratories across 15 NIH institutes and centers, culminating in publication of over 230 peer-reviewed articles in highly acclaimed biomedical and biological scientific journals. Over the past 12 months, the Facility supported the research needs of 141 investigators, fellows, students, and laboratory staff contributing to 133 basic science, clinical and translational research projects in 32 NINDS and other intramural laboratories and facilitated multidisciplinary collaborative projects between NINDS and 6 other NIH institutes and centers. Contributions by this Facility over the past 12 months were incorporated in publications of 16 peer-reviewed articles in highly acclaimed scientific journals and expedited the preparation of 18 more articles currently in preparation for journal submission or in journal peer review.

NINDS Flow and Imaging Cytometry Core Facility（Facility）由NINDS的研究人员于2001年建立，作为NINDS和其他内部研究人员的重要资源，以支持他们在基础和临床研究项目中的工作。该设施不断提供由该设施管理的流式细胞仪和成像设备的无缝使用，以及培训调查人员正确和最佳使用这些设备的关键专门知识。该机构的工作人员还可以使用适当的细胞表面、细胞质、细胞核、蛋白质、基因表达和生理指示剂染料生物标志物的独特组合，为设计和优化各种新型和定制的多重染色方案提供建设性意见，这些生物标志物对任何特定项目的启动研究者都特别感兴趣。根据要求，机构工作人员定期进行多色流式细胞术和原位细胞术实验，并分析来自单个或多个感兴趣细胞群的数据。应用基于测试的生物标志物的不同组合的用户定义的布尔逻辑门控策略，该设施向每个研究者提供感兴趣的单个细胞或细胞群中复杂信号分布的定量说明和解释。使用流式细胞仪的制备细胞分选能力，机构工作人员可根据启动研究者的要求，协助从异质细胞制备物中分离高度纯化的细胞群和亚群。这些包括从CNS和PNS组织中分选神经干细胞和祖细胞、特定神经元和神经胶质细胞类型以及非神经细胞类型（小胶质细胞、内皮细胞）。此外，该机构还对来自骨髓、外周血、脾脏和脑脊液的非CNS来源干细胞（造血、间充质和Hoechst染料排除侧群）和不同白细胞表型（淋巴细胞、单核细胞、中性粒细胞、嗜碱性粒细胞、嗜酸性粒细胞、肥大细胞）进行常规分类。然后，NINDS和其他内部研究人员常规使用这些高度纯化的感兴趣的细胞进行进一步的体外和体内研究，包括细胞培养、移植、延时和生理指示剂染料成像，以及用感兴趣的特定基因构建体进行转导或转染。此外，FACS纯化的细胞已被用作蛋白质印迹和免疫沉淀实验的宝贵蛋白质来源以及RT-PCR、微阵列、RNA-Seq和microRNA测定的RNA来源。这些细胞还提供了前所未有的见解，具体的功能丧失实验使用反义和siRNA探针。在上述应用中使用FACS纯化的同质细胞的主要意义在于，这种方法减少了研究异质细胞制备物时固有存在的发现中的模糊性。总而言之，定量流式细胞术和原位细胞仪分析的常规应用以及该设施提供的制备细胞分选服务为NINDS和其他内部研究人员提供了许多细胞生物学方面的见解，这些见解无法以其他方式得到证明。自2001年成立以来，该基金已为500多名NINDS和其他校内研究人员，研究员，学生和科研人员提供了这些宝贵的服务，并支持了来自15个NIH研究所和中心的105个实验室的700多个基础，转化和临床研究项目的多学科科学研究。最终在备受赞誉的生物医学和生物科学期刊上发表了230多篇同行评审的文章。在过去的12个月里，该基金支持了141名研究人员，研究员，学生和实验室工作人员的研究需求，为32个NINDS和其他校内实验室的133个基础科学，临床和转化研究项目做出了贡献，并促进了NINDS和其他6个NIH研究所和中心之间的多学科合作项目。该机制在过去12个月里提供的资料被纳入在备受赞誉的科学期刊上发表的16篇同行审查文章，并加快了目前正在准备提交期刊或期刊同行审查的另外18篇文章的编写工作。